Several techniques have been developed thus far to transfer DNA into cells, but none demonstrate real satisfaction. One of them, derived from the Mandel and Higa technique, based on acquisition by E. coli susceptible to transformation by treatment with CaCl2, consists of co-precipitating DNA with calcium phosphate or DEA dextran, and introducing the precipitate obtained directly into the cell or nucleus. This method, besides the fact that it is toxic, is not selective.
Another method of directly introducing DNA, electroporation, consists of giving the cells a brief electric shock of several thousand volts, which allows the DNA to pass through the cytoplasmic membrane and to be introduced into the cell. This method is very toxic to the cells, involving high mortality and great variability, depending on the cells used. Other methods use receptors on the membrane to screen the entry of the gene into the cells. The DNA can then penetrate into the cell through either a specific ligand of these receptors, or specific antibodies of membrane constituents. The DNA-ligand complex thus penetrates into the cell by a process of endocytosis. This process is limited due to the fact that there is major destruction of the complex used in the lysosomal vesicles. Several processes have been developed to eliminate these disadvantages, but none gives complete satisfaction.
U.S. Pat. No. 5,635,383 describes another type of complex vector, based on polylysine, for transferring nucleic acids into cells.
U.S. Pat. No. 5,521,291 describes another method based on the use of a conjugate formed from a virus linked to a substance having a strong affinity for DNA via an antibody. Such conjugates are hard to use, and certain risks are associated with the use of viruses.
To try to eliminate these disadvantages, a process was described in patent application No. WO 97/02840 that is used in vitro and consists of using anti-DNA murine antibodies, or their F(ab′)2 and Fab′ fragments, capable of penetrating inside live cells, as immunovectors for intracytoplasmic and/or intranuclear transfer of biologically active substances. Although these vectors are highly effective, they can be complicated to use in some applications. Using molecules the size and complexity of antibodies can also be a major disadvantage in their handling and implementation.
Patent application No. WO 99/07414 described how it was possible to use peptides in vitro, derived from anti-DNA murine antibodies, described in the above-mentioned application WO 97/02840, as intracytoplasmic and intranuclear internalization vectors for biologically active substances.
Although these murine-origin peptide vectors are coded by the germinal line and carry no mutation, and consequently, should be antigenically close to those encountered in humans, the risk of an immune reaction in humans cannot be excluded.
There is a need for peptides and amino acid sequences that eliminate the disadvantages described above, namely that can be used as, or in, a cell internalization vector in humans and would pose none of the risks mentioned above. Having tools that make it possible to transfer substances of interest effectively from the outside medium to the inside of cells, and more specifically cell nuclei, is a major advantage in the field of biotechnology, especially to produce proteins or peptides to regulate the expression of genes, to analyze and screen intracellular signaling paths, or to analyze the properties of a given substance on a cell. Another important application of such tools concerns the field of gene therapy since, up until the present time, the various gene therapy processes have run into the same need, not met in an optimal way, which is to be able to have vectors that can transfer biologically active ingredients into the cytoplasm and/or the cell nucleus from the host organism being treated, without thereby altering either the genome of the host or the biological properties of the active ingredients transferred.